Metallothionein-Derived Peptide Fragments

ABSTRACT

The present invention relates to neural cell survival, differentiation and proliferation promoting peptide fragments derived from metallothioneins (MT), pharmaceutical compositions comprising said peptide fragments and uses thereof for treatment of diseases and conditions where the effects of stimulating neural cell proliferation, differentiation and/or survival, and/or stimulating neural plasticity associated with learning and memory are beneficial for treatment.

FIELD OF INVENTION

The present invention relates to neural cell survival, differentiationand proliferation promoting peptide fragments derived frommetallothioneins (MT), pharmaceutical compositions comprising saidpeptide fragments and uses thereof for treatment of diseases andconditions where the effects of stimulating neural cell proliferation,differentiation and/or survival, and/or stimulating neural plasticityassociated with learning and memory are beneficial for treatment.

BACKGROUND OF INVENTION

Metallothioneins (MTs) are a class of ubiquitously occurring lowmolecular weight cysteine- and metal-rich proteins containingsulfur-based metal clusters. The conservation of these clusters in anincreasing number of three-dimensional structures of invertebrate,vertebrate and bacterial MTs signifies the importance of this structuralmotif. It is becoming increasingly clear that mammalian MTs have diversefunctions including involvement in zinc homeostasis, protection againstheavy metal toxicity and oxidative damage (Vasak et al., 2005).Mammalian MTs are single chain polypeptides of 61, 60 or 68 amino acidresidues with an N-terminal acetylmethionine and often alanine at thecarboxyl terminus. They contain 20 cysteine residues, which are centralto the binding of metals. MTs have characteristic C-X-C, C-Y-C, and C-Csequences, where X and Y are non-cysteine amino acids. There are 7bivalent ions for every 20 cysteines forming metal thiolate complexes(7-10 g atoms of metal per mol MT in a two domain structure (Hussain etal., 1996).

There are four MT subgroups, namely MT1, MT2, MT3, and MT4. The MT1 andMT2 isoforms, which differ by only a single negative charge, are themost widely expressed isoforms in different tissues. Human MT genes areclustered at a single locus on chromosome 16, and at least 14 of the 17genes so far identified, are functional. These encode multiple isoformsof MT1 (MT1A, B, E, F, G, H, I, K, L and X), MT2, MT3 and MT4 (Miles etal., 2000).

Stimuli that can induce MT expression are metals. hormons (e.g.glucocorticoids), cytokines, a variety of other chemicals, inflammation,and stress. MT degradation takes place mainly in the lysosomes. MTappears less susceptible to proteolysis in the metal bound state. Invivo, metal-MTs have far longer half-lives than apo-MT (Miles et al.,2000).

MT1 and MT2 are present throughout the brain and spinal cord, and thatthe main cell type expressing these MT isoforms is the astrocyte;nevertheless, MT1 and MT2 expression was also found in ependymal cells,epithelial cells of choroid plexus, meningeal cells of the pia mater,and endothelial cells of blood vessels (Hidalgo et al., 2001).

MTs are stress-inducible proteins that maintain metal homeostasis andscavenge free radicals. It is generally accepted that the majorfunctions of MTs are related to metal metabolism. Postulated functionsinclude detoxification and storage of heavy metals and the regulation ofcellular copper and zinc metabolism in response to dietary andphysiological changes. Because astrocytes, as well as ependymal cells,richly express MTs, an attractive hypothesis is that both cell typesserve to protect the CNS from metals transported parenchymally from theblood or the cerebrospinal fluid. In AD subjects cerebral white mattercontains numerous MT1- and MT2-expressing astrocytes with an intenseimmunoreactivity of the cell body (Zambenedetti et al., 1998). Chronicinflammation has been postulated raising the possibility that theetiology of AD has an immunological component. Cytokines and interleukin(IL)-1, for instance, elevated in AD, induce MT1 and MT2 production inastrocytes suggesting that these proteins may have a relevant role inproviding long-term protection against oxidative damage, injury andinflammation with a multiple compensatory mechanism involving theosmotic regulation of some metal ions. Clear-cut effects of CNS injuryon MT1 and MT2 expression were investigated. These studies have shown adramatic induction of these MT isoforms in response to kainicacid-induced seizures, cryogenic injury, ischaemia, and after treatmentwith 6-aminonicotinamide (Penkowa et al., 1995; 2000; 2001). MT1 and MT2are significant inhibitors of apoptotic cell death in the CNS (Giralt etal., 2002). MT1 and MT2 deficient mice showed both increased oxidativestress and neuronal apoptosis during epileptic seizures, experimentalautoimmune encephalomyelitis (EAE), and following traumatic braininjury. Likewise, transgenic MT1 overexpressing mice showedsignificantly reduced oxidative tissue damage and cell death duringtraumatic brain injury, focal cerebral ischemia, and 6-aminonicotinamide(6-AN)-induced brain stem toxicity. Furthermore, MT1 and MT21 improvethe clinical outcome and reduce mortality in different CNS disorders(Penkowa, 2002). MT has recently been shown to mediate neuroprotectionin genetically engineered mouse model of Parkinson's disease (Ebadi etal., 2005).

MT2 treatment has recently been shown to significantly stimulate neuriteextension from both dopaminergic and hippocampal neurons. Moreover, MT2treatment significantly increases survival of dopaminergic neuronsexposed to 6-hydroxydopamine (6-OHDA) and protects significantlyhippocampal neurons from amyloid β-peptide-induced neurotoxicity (Køhleret al., 2003). Treatment using MT2 and other MTs has been suggested formotor neuron disease, head injury, Alzheimer's and Parkinson's diseases(WO03105910). The molecular mechanisms of neuritogenic andneuroprotective actions of MTs are so far unknown.

Recently, it has been shown that MT1 binds to low-density lipoproteinreceptor related protein 2 (LRP2)/megalin and the corresponding bindingsite in MT1 has been identified (Klassen et al., 2004).

Megalin/LRP2 is a scavenger receptor due to its multifunctional bindingproperties. Among its ligands are lipoproteins, vitamin-binding andcarrier proteins, drugs, hormones and enzymes as well as signallingmolecules. The intracellular domain of megalin interacts with signallingadaptor molecules which has been shown to be involved in regulation ofedocytosis (see for review May et al, 2005). However, it remains to beuncertain, whether megalin participates directly in cellular signallingcascades by transducing extracellular signals to intracellular bindingpartners.

One of the best-characterized physiological functions of megalin is theproximal-tubular reuptake of low-molecular weight proteins (Zou et al.,2004). Another permanent feature is that megalin is required for aproper forebrain development: megalin knock-out mice demonstrateholoprosencephaly (Willnow et al., 1996).

REFERENCES

-   Vasak M. Advances in metallothionein structure and functions. J    Trace Elem Med Biol. 2005, 19:13-17.-   Giralt M, Penkowa M, Lago N, Camats J, Hernandez J, Molinero A and    Hidalgo J. Metallothionein-1+2 protect the CNS after a focal brain    injury. Exp. Neurol. 2002, 173pp: 114-128.-   Ebadi M, Brown-Borg H, El Refaev H, Singh B B, Garret S, Shavali S    and Sharma S K. Metallothionein-mediated neuroprotection in    genetically engineered mouse models of Parkinson's disease. 2005,    134:67-75.-   Hidalgo H, Aschner M, Zatta P, and Vasak M. Roles of the    metallothionein family of proteins in the central nervous system.    Brain Res Bull. 2001, 55:133-145.-   Hussain S, Slikker W, and Ali S F. Role of metallothioneins and    other antioxidants in scavenging superoxide eadicals and their    possible role in neuroprotection, Neurochem. Int. 1996, 29:145-152.-   Klassen R B, Crenshaw K, Kozyraki R, Verroust P J, Tio L, Atrian S,    Allen P L, Hammond T G Megalin mediates renal uptake of heavy metal    metallothionein complexes. Am J Physiol Renal Physiol. 2004,    287:F393-403.-   Køhler L B, Berezin V, Bock E and Penkowa M. The role of    metallothionein II in neuronal differentiation and survival. Brain    Res. 2003, 992:128-136.-   May P, Herz J, Bock H H. 2005 Molecular mechanisma of lipoprotein    receptor signalling. Cell Moll Life Sci. 62:2325-2338.-   Miles A T, Hawksworth G M, Beattie J H, Rodilla V. Induction,    regulation, degradation, and biological significance of mammalian    metallothioneins. Crit Rev Biochem Mol Biol. 2000, 35:35-70.-   Penkowa M and Moos T. Disruption of the blood-brain interface in    neonatal rat neocortex induces a transient expression of    metallothionein in reactive astrocytes. Glia 1995, 13:217-227.-   Penkowa M and Hidalgo J, Metallothionein I+II expression and their    role in experimental encephalomyelitis. Glia 2000, 32:247-263.-   Penkowa M, Giralt M, Carrasco J, Hadberg H and Hidalgo H. Impaired    inflammatory response and increased oxidative stress and    neurodegeneration after brain injury in interleukin-6-deficient    mice. Glia 2000, 32:271-285.-   Penkowa M Giralt M, Thomsen P, Carrasco J and Hidalgo J. The zinc or    copper deficiency-induced impaired inflammatory response to brain    trauma may be caused by the concomitant metallothionein changes. J.    Neurotrauma 2001, 18:447-463.-   Penkowa M. Metallothionein expression and roles in the central    nervous system. Biomed. Rev. 2002, 13:1-18.-   Rønn L C, Ralets I, Hartz B P, Morten B, Berezin A, Berezin V,    Møller A, and Bock E. A simple procedure for quantification of    meurite outgrowth based on stereological principles. J. Neurosci.    Meth. 2000, 100:25-32.-   Tang W, Kido T, Gross W A, Nogawa K, Sabbioni E and Shaikh Z A.    Measurement of cadmium-induce metallothionein in urine by ELISA and    prevention of overestimation due to polymerization. J. Anal.    Toxicol. 1999, 23:153-158.-   Willnow T E, Hilpert J, Armstrong S A, Rohlmann A, Hammer A E, Burns    D K, et al. 1996. Defective forebrain development in mice lackin    gp339/megalin. Proc Natl Acad Sci USA 93:8460-8464.-   Zambenedetti P, Giordano R and Zatta P. Metallothioneins are highly    expressed in astrocytes and microcapillaries in Alzheimer's    disease. J. Chem. Neuroanat. 1998, 15:21-26.-   Zou Z, Chung B, Nguyen T, Mentone S, Thompson B, and Biemesderfer D.    2004 Linking receptor-mediated endocytosis and cell signalling:    evidence for regulated intramembrane proteolisis of megalin in    proximal tubule. J Biol Chem 179:34302-34310.

SUMMARY OF THE INVENTION

The present invention relates to short peptide sequences capable ofstimulating neuronal cell differentiation, neuronal cell survival andneural plasticity associated with learning and memory, capable ofinhibiting the oxidative stress and inflammatory responses, modulatingof survival and neuroprotection, promoting the effects of growth factorsand regulatory molecules and their receptors.

According to one aspect of the invention, the peptide sequencesdescribed herein comprise a common structural motif which is essentialfor biological activity of peptides.

Such peptides according to the invention comprise a sequence of at most25 contiguous amino acid residues which comprise an amino acid motif ofthe formula: S/D/E-(x)_(n)-S/D/E-K/S, wherein (x)_(n) is a sequence ofany amino acid residues with an integer n from 4 to 6.

A peptide as above is according to the invention homologous to asubsequence of a protein of the metallothionein family, ametallothionein (MT), wherein said subsequence is a functional domain ofMT involved in stimulation of neurite outgrowth, neural cells survival,neural plasticity associated with learning and memory and/or inhibitionof oxidative stress, inflammatory responses by MT, and it is alsoinvolved in homodimerization (homophylic binding) of metallothioneins.Accordingly, a peptide sequence comprising the amino acid motif of theinvention is not only a structural homologue the latter MT functionaldomain, but as well a functional homologue of MT protein or a peptidefragment of MT derived from MT functional domain involved in executionof the mentioned functions of MT.

Accordingly, the invention in another aspect relates to a peptidefragment of MT capable of mimicking biological function of MT, forexample capable of stimulating neurite outgrowth, neuronal survival,neural plasticity associated with learning and memory and/or inhibitinginflammation. Still, in another aspect, the invention relates to afragment of MT which is capable of modulating of biological function ofMT by stimulating or inhibiting homophylic binding of MT or bystimulating or inhibiting MT binding to its receptor.

The invention also relates to a compound comprising a peptide sequencecomprising the motif of the invention and/or peptide fragment of MT.

Further aspects of the invention relate

-   -   to use of a peptide sequence of the invention as a medicament or        for the manufacturing of a medicament, said medicament is for        treatment a condition involving stimulating neurite outgrowth,        neural cell survival, neural cell plasticity associated with        learning and memory and/or inhibiting inflammation;    -   pharmaceutical composition comprising a peptide sequence of the        invention or compound comprising thereof;    -   antibody capable of binding to an epitope comprising a peptide        sequence of the invention;    -   pharmaceutical composition comprising an antibody of the        invention;    -   methods of treatment involving using peptide sequences,        compounds, medicaments, antibodies of the invention or        pharmaceutical compositions comprising thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Effect of the MTAc peptide on neurite outgrowth

FIG. 2 Effect of the MTBc peptide on neurite outgrowth.

FIG. 3 Effect of the MTAcc peptide on neurite outgrowth.

FIG. 4 Effect of the MTBcc peptide on neurite outgrowth.

FIG. 5 Effect of the MTBnc peptide on neurite outgrowth.

FIG. 6 Effect of the MTAc peptide on neuronal survival.

FIG. 7 Effect of the MTBc peptide on neuronal survival.

FIG. 8 Effect of the MTBn peptide on neuronal survival.

FIG. 9 Effect of the MTBcc, MTAcc, MTBnc and MT31p peptides on neuronalsurvival.

FIG. 10 Effect of the MTAcc and MTBcc peptides on DNA fragmentation.

FIG. 11 Effect of the MTAcc peptide on neuronal survival in vivodemonstrated as the counts of cells demonstrating immunostaining forselected markers of inflammation (IL-1, IL12 and THFα), oxidative stress(NITT and MDA) and apoptotic cell death (TUNEL)

FIG. 12 Binding of the MTAcc peptide to MT studied by means of SPRanalysis. Approximately 2000 resonance units (RU) of the MT2 protein(Sigma) were immobilized on the sensor chip. The binding is given as theresponse difference between the binding to the sensor chip with theimmobilized MT2 and a blank sensor chip (unspecific binding). Thepeptide was injected into the sensor chip at a concentration of 0.87 μM.The experiment was repeated four times.

FIG. 13 Binding of the MTBcc peptide to MT studied by means of SPRanalysis. Approximately 2000 resonance units (RU) of the MT2 protein(Sigma) were immobilized on the sensor chip. The binding is given as theresponse difference between the binding to the sensor chip with theimmobilized MT2 and a blank sensor chip (unspecific binding). Thepeptide was injected into the sensor chip at a concentration of 8.32 μM.The experiment was repeated four times.

FIG. 14 Binding of the MTAn peptide to MT studied by means of SPRanalysis. Approximately 2000 resonance units (RU) of the MT2 protein(Sigma) were immobilized on the sensor chip. The binding is given as theresponse difference between the binding to the sensor chip with theimmobilized MT2 and a blank sensor chip (unspecific binding). Thepeptide was injected into the sensor chip at a concentration of 1.7 μM.

DETAILED DESCRIPTION OF THE INVENTION 1. Peptide

In a first aspect the present invention relates to a peptide comprisingof at most 25 contiguous amino acid residues comprising an amino acidmotif of the formula: S/D/E-(x)_(n)-S/D/E-K/S, wherein (x)_(n) is asequence of any amino acid residues with an integer n from 4 to 6.

In one embodiment, (x)n is an amino acid sequence wherein n is 4, inanother embodiment n is 6. The amino acid sequence (x)n is a sequence ofany amino residues, however, in some preferred embodiments it maycomprise at least one of the following amino acid residues: K, S, E orC.

The invention preferably relates to peptide sequences comprising aminoacid residues in the range of 8-25 amino acid residues, such as from 9to 25 amino acid residues, for example from 10 to 25 amino acidresidues. In some embodiments the peptide sequence may be between 11 and25 amino acid residues, such as between 12 and 25, for example from 13to 25 amino acid residues. In other embodiments the amino acid sequencemay comprise about 15 amino acid residues, such as 14 or 16 amino acidresidues, or it may be about 20 amino acid residues in length, such asfrom 17 to 19 amino acid residues, or it may comprise from 20 to 25amino acid residues. A short amino acid sequence comprising at most 20contiguous amino acid residues is preferred, however, the inventionincludes in the scope amino acid sequences which may comprise about 23or 25 amino acid residues, such as 21, 22, 23, 24, 25 or 26 amino acidresidues, or may be about 30 amino acid residues, such as 27, 28 or 29amino acid residues, or be about 35 amino acid residues, such as from 31to 34 amino acid residues.

A peptide comprising the motif of the invention, may comprise in someembodiments at least one amino acid residue G within a sequence of 10amino acid residues comprising the motif, preferably, amino acid residueG precedes the amino acid residue S/D/E at any corresponding position ofthe motif.

The invention also relates to a peptide as above wherein the amino acidresidue S/D/E at any position of the motif is substituted for amino acidresidue C.

A peptide as above may for example comprise or consist of an amino acidsequence selected from the following sequences:

(SEQ ID NO: 1) KKSSCSCSPVGSAK (SEQ ID NO: 2) AQGSISKGASDKSS(SEQ ID NO: 3) MDPNSSSAAGDSST (SEQ ID NO: 4) SAGSSKSKESKSTS(SEQ ID NO: 5) AQGSICKGASDKSS (SEQ ID NO: 6) MDPNCSCAAGDSST(SEQ ID NO: 7) SAGSCKCKESKSTS (SEQ ID NO: 8) KGGEAAEAEAEK,or be a fragment, or a variant of any of these sequences.

According to the invention a peptide comprising a sequence selected fromSEQ ID NOS:1-8 is homologous to a subsequence of a metallothioneinselected from the group consisting of metallothionein-1 A (MT1A),metallothionein-1B (MT1B), metallothionein-1E (MT1E), metallothionein-1F(MT1F), metallothionein-1G (MT1G), metallothionein-1H (MT1H),metallothionein-1I (MT1I), metallothionein-1 K (MT1K), metallothionein-1L (MT1L), metallothionein-1R (MT1R), metallothionein-1X (MT1 X),metallothionein-2 (MT2), metallothionein-3 (MT3) and metallothionein-4(MT4). The sequences of the latter mentioned metallothioneins areidentified in the Gene Bank under the following Acc. Nos: Q9BQN2,P04731, P07438, P04732, P04733, P13640, P80294, P80295, P80296, Q93083,P80297, P02795, P25713, P47944, respectively.

More particular, a peptide comprising a sequence selected from SEQ IDNOs: 1-8 is homologous to a subsequence of MT which comprises one of thefollowing amino acid sequences:

(SEQ ID NO: 9) KKSCCSCCPMSCAK (SEQ ID NO: 10) KKCCCSCCPVGCAK(SEQ ID NO: 11) KKSCCSCCPVGCAK (SEQ ID NO: 12) KKSCCSCCPVGCSK(SEQ ID NO: 13) KKSCCSCCPVGCAK (SEQ ID NO: 14) KKSCCSCCPLGCAK(SEQ ID NO: 15) KKSCCSCCPVGCAK (SEQ ID NO: 16) KKSCCSCCPVGCAK(SEQ ID NO: 17) KKSCCSCCPVGCAK (SEQ ID NO: 18) KKSCCSCCPMGCAK(SEQ ID NO: 19) KKSCCSCCPVGCAK (SEQ ID NO: 20) KKSCCSCCPVGCAK(SEQ ID NO: 21) KKSCCSCCPAECEK (SEQ ID NO: 22) RKSCCPCCPPGCAK(SEQ ID NO: 23) AQGCICKGASEKCS (SEQ ID NO: 24) AQGCVCKGSSEKCS(SEQ ID NO: 25) AQGCVCKGASEKCS (SEQ ID NO: 26) AQGCVCKGASEKCS(SEQ ID NO: 27) AQGCICKGASEKCS (SEQ ID NO: 28) AQGCICKGASEKCS(SEQ ID NO: 29) AQGCICKGASEKCS (SEQ ID NO: 30) AQGCICKGASEKCS(SEQ ID NO: 31) AQGCICKGTSDKCS (SEQ ID NO: 32) AQGCVCKGASEKCS(SEQ ID NO: 33) AQGCICKGTSDKCS (SEQ ID NO: 34 AQGCICKGASDKCS(SEQ ID NO: 35) AKDCVCKGGEAAEAEAEKCS (SEQ ID NO: 36) ARGCICKGGSDKCS(SEQ ID NO: 37) MDPNCSCATGGSCT (SEQ ID NO: 38) MDPNCSCTTGGSCA (SEQ ID NO: 39) MDPNCSCATGGSCT (SEQ ID NO: 40) MDPNCSCAAGVSCT(SEQ ID NO: 41) MDPNCSCAAGVSCT (SEQ ID NO: 42) MDPNCSCEAGGSCA(SEQ ID NO: 43) MDPNCSCAAGVSCT (SEQ ID NO: 44) MDPNCSCAAAGVSCT(SEQ ID NO: 45) MDPNCSCSPVGSCA (SEQ ID NO: 46) MDPNCSCATGGSCS(SEQ ID NO: 47) MDPNCSCDPVGSCA (SEQ ID NO: 48) MDPNCSCAAGDSCT(SEQ ID NO: 49) MDPETCPCPSGGSCT (SEQ ID NO: 50) MDPRECVCMSGGICM(SEQ ID NO: 51) CTGSCKCKECKCNS (SEQ ID NO: 52) CAGSCKCKECKCTS(SEQ ID NO: 53) CAGSCKCKECKCTS (SEQ ID NO: 54) CAGSCKCKECKCTS(SEQ ID NO: 55) CASSCKCKECKCTS (SEQ ID NO: 56) CAGSCKCKKCKCTS(SEQ ID NO: 57) CAGSCKCKECKCTS (SEQ ID NO: 58) CASSCKCKECKCTS(SEQ ID NO: 59) CAGSCKCKECKCTS (SEQ ID NO: 60) CASSCKCKECKCTS(SEQ ID NO: 61) CAGSCKCKECKCTS (SEQ ID NO: 62) CAGSCKCKECKCTS(SEQ ID NO: 63) CADSCKCEGCKCTS (SEQ ID NO: 64) CGDNCKCTTCNCKT.

In different embodiments of the invention it may preferably be selecteda peptide which is homologues to a specific subsequence of a selectedgroup of subsequences. Accordingly, in one embodiment it may be apeptide which is homologues to a subsequence which is selected from thegroup of subsequences identified as SEQ ID NOs:9-22.

In another embodiment it may be a peptide which is homologous to asubsequence comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:23-36.

In other embodiments it may be a peptide which is homologues to asubsequence which comprise an amino acid sequence selected from any ofthe sequences identified as SEQ ID NOs:37-50, or to a a subsequencewhich comprise an amino acid sequence selected from any of the sequencesidentified as SEQ ID NOs: 51-64.

Still in other embodiments, a peptide may be homologues to a subsequenceof a particular MT, for example to be homologous to a subsequence ofMT1A, said subsequence is selected from SEQ ID NOs:9, 23, 37 or 51; or asubsequence of MT1B, said subsequence is selected from SEQ ID NOs:10,24, 38 or 52. The following are other examples of preferred subsequencesof particular MTs which are included in the scope of the invention:

-   -   MT1E subsequence selected from SEQ ID NOs:11, 25, 39 or 53;    -   MT1F subsequence selected from SEQ ID NOs:12, 26, 40 or 54;    -   MT1G subsequence selected from SEQ ID NOs:13, 27, 41 or 55;    -   MT1H subsequence selected from SEQ ID NOs:14, 28, 42 or 56;    -   MT1I subsequence selected from SEQ ID NOs:15, 29, 43 or 57;    -   MT1K subsequence selected from SEQ ID NOs:16, 30, 44 or 58;    -   MT1L subsequence selected from SEQ ID NOs:17, 31, 45 or 59;    -   MT1R subsequence selected from SEQ ID NOs:18, 32, 46 or 60;    -   MT1X subsequence selected from SEQ ID NOs:19, 33, 47 or 61;    -   MT2 subsequence selected from SEQ ID NOs:20, 34, 48 or 62;    -   MT3 subsequence selected from SEQ ID NOs:21, 35, 49 or 63;    -   MT4 subsequence selected from SEQ ID NOs:22, 36, 50 or 64.

Any of the subsequences of the MTs identified above may also be a partof an amino acid sequence of the peptide according to invention. Thus, afurther aspect of the invention relates to a peptide comprising an aminoacid sequence selected from SEQ ID NOs:9-64, or comprising a fragment orvariant of said sequence, or consisting of any of these sequences,fragments or variants.

Sequence homology may be calculated using well known algorithms such asBLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90.The terms “sequence similarity” sequence identity” and “sequencehomology” are used in the present application interchangeably whenreferred to a number or percentage of identical or similar amino acidresidues in two collated amino acid sequences. “Similar amino acidresidues” are amino acid residues derived from the same group of“conservative” amino acid residues. The latter groups are discussedfurther in the application.

In the present application the standard one-letter code for amino acidresidues is applied as well as the standard three-letter code.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide of the invention exists as the free carboxylicacid, this may also be specified as “—OH”. However, the C-terminal aminoacid of a compound of the invention may be the amidated derivative,which is indicated as “—NH₂”. Where nothing else is stated theN-terminal amino acid of a polypeptide comprise a free amino-group, thismay also be specified as “H—”.

Where nothing else is specified amino acid can be selected from anyamino acid, whether naturally occurring or not, such as alfa aminoacids, beta amino acids, and/or gamma amino acids. Accordingly, thegroup comprises but are not limited to: Ala, Val, Leu, Ile, Pro, Phe,Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, HisAib, Nal, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Also, according to the invention modifications of the compounds/peptidesmay be performed, such as for example glycosylation and/or acetylationof the amino acids.

Basic amino acid residues are according to invention represented by theresidues of amino acids Arg, Lys, and His, acidic amino acid residues—bythe residues of amino acids Glu and Asp. Basic and acidic amino acidresidues constitute a group of charged amino acid residues. The group ofhydrophobic amino acid residues is represented by the residues of aminoacids Leu, Ile, Val, Phe, Trp, Tyr, Met, Ala and Pro.

The invention relates to naturally occurring, synthetically/recombinantprepared peptide sequence/fragments, and/or peptide sequence/fragmentsprepared by means of enzymatic/chemical cleavage of a biggerpolypeptide, wherein said peptide sequence/fragments are integral partsof said bigger polypeptides. The invention relates to isolatedindividual peptide sequences.

As it is mentioned above, the invention relates to variants of peptidesequences described in the application as well.

In one aspect the term “variant of a peptide sequence” means that thepeptides may be modified, for example by substitution of one or more ofthe amino acid residues. Both L-amino acids and D-amino acids may beused. Other modification may comprise derivatives such as esters,sugars, etc. Examples are methyl and acetyl esters.

In another aspect “variants” may be understood as exhibiting amino acidsequences gradually differing from the preferred predetermined sequence,as the number and scope of insertions, deletions and substitutionsincluding conservative substitutions increase. This difference ismeasured as a reduction in homology between the predetermined sequenceand the variant.

In still another aspect, variants of the peptide fragments according tothe invention may comprise, within the same variant, or fragmentsthereof or among different variants, or fragments thereof, at least onesubstitution, such as a plurality of substitutions introducedindependently of one another. Variants of the complex, or fragmentsthereof may thus comprise conservative substitutions independently ofone another, wherein at least one glycine (Gly) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Ala, Val, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone alanine (Ala) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Val, Leu, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one valine (Val) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Gly, Ala, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone leucine (Leu) of said variant, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Ala, Val, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one isoleucine (Ile) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Val andLeu, and independently thereof, variants, or fragments thereof whereinat least one aspartic acids (Asp) of said variant, or fragments thereofis substituted with an amino acid selected from the group of amino acidsconsisting of Glu, Asn, and Gln, and independently thereof, variants, orfragments thereof, wherein at least one aspargine (Asn) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Gln,and independently thereof, variants, or fragments thereof, wherein atleast one glutamine (Gin) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Asn, and wherein at least one phenylalanine(Phe) of said variants, or fragments thereof is substituted with anamino acid selected from the group of amino acids consisting of Tyr,Trp, His, Pro, and preferably selected from the group of amino acidsconsisting of Tyr and Trp, and independently thereof, variants, orfragments thereof, wherein at least one tyrosine (Tyr) of said variants,or fragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Trp, His, Pro, preferably anamino acid selected from the group of amino acids consisting of Phe andTrp, and independently thereof, variants, or fragments thereof, whereinat least one arginine (Arg) of said fragment is substituted with anamino acid selected from the group of amino acids consisting of Lys andHis, and independently thereof, variants, or fragments thereof, whereinat least one lysine (Lys) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Arg and His, and independently thereof, variants, orfragments thereof, and independently thereof, variants, or fragmentsthereof, and wherein at least one proline (Pro) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Tyr, Trp, and His, andindependently thereof, variants, or fragments thereof, wherein at leastone cysteine (Cys) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofAsp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

It thus follows from the above that the same functional equivalent of apeptide fragment, or fragment of said functional equivalent may comprisemore than one conservative amino acid substitution from more than onegroup of conservative amino acids as defined herein above. The term“conservative amino acid substitution” is used synonymously herein withthe term “homologous amino acid substitution”.

The groups of conservative amino acids are as the following:

P, A, G (neutral, weakly hydrophobic),S, T (neutral, hydrophilic)Q, N (hydrophilic, acid amine)E, D (hydrophilic, acidic)H, K, R (hydrophilic, basic)L, I, V, M, F, Y, W (hydrophobic, aromatic)C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide of the invention or fragment thereof. Itmay however also be desirable to introduce non-conservativesubstitutions, particularly, but not limited to, a non-conservativesubstitution in any one or more positions. In particular, a variantwhich may be an amino acid sequence having at least 60%, more preferablyat least 70%, more preferably at least 80%, more preferably at least90%, more preferably 95% homology to an amino acid sequence of theinvention, such as a sequence comprising the motif of the invention, forexample a sequence selected from SEQ ID NOs: 1-64, or may be an aminoacid sequence having at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferably95% positive amino acid matches compared to an amino acid sequence ofthe invention, such as a sequence comprising the motif of the invention,for example a sequence selected from SEQ ID NOs: 1-64. A positive aminoacid match is defined herein as an identity or similarity defined byphysical and/or chemical properties of the amino acids having the sameposition in two compared sequences. Preferred positive amino acidmatches of the present invention are K to R,

E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T,N to S and Q to R. The homology of one amino acid sequence with anotheramino acid is defined as a percentage of identical amino acids in thetwo collated sequences. The homology of the sequences as mentioned abovemay be routinely calculated using well known algorithms such as BLOSUM30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62,BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of the peptide of the invention wouldfor example differ substantially in polarity, for example a residue witha non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)substituted for a residue with a polar side chain such as Gly, Ser, Thr,Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, orLys, or substituting a charged or a polar residue for a non-polar one;and/or ii) differ substantially in its effect on peptide backboneorientation such as substitution of or for

Pro or Gly by another residue; and/or iii) differ substantially inelectric charge, for example substitution of a negatively chargedresidue such as Glu or Asp for a positively charged residue such as Lys,His or Arg (and vice versa); and/or iv) differ substantially in stericbulk, for example substitution of a bulky residue such as His, Trp, Pheor Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and viceversa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like. Exemplary amino acid substitutions which takevarious of the foregoing characteristics into consideration are wellknown to those of skill in the art and include: arginine and lysine;glutamate and aspartate; serine and threonine; glutamine and asparagine;and valine, leucine and isoleucine.

The following variants are preferred by the invention:

-   1. a variant which is an amino acid sequence of at least 6 amino    acid residues having at least 65% sequence similarity with a    sequence selected from the sequences of SEQ ID NOs:1-64, preferably    an amino acid sequence of 6 to 20 contiguous amino acid residues,    which has more then 70% sequence similarity with a sequence selected    from the sequences of SEQ ID NOs:1-64, such as from 71% to 80%    similarity, preferably from 81% to 85%, more preferably from 86% to    90%, even more preferably from 91% to 95%, and even more preferably    more then 95% of sequence similarity.-   2. a variant which consists of a sequence of SEQ ID NOs:1-64, said    sequence comprising one or more modifications of amino acid    residues, such as for example modification as discussed above.

As it is mentioned above, the present invention also relates tofragments of the peptide sequences described in the application.

A preferred fragment of the invention is a fragment of a sequenceselected from SEQ ID NOs:1-64 which has the length of about 40% of thelength of said sequence, more preferably at least 50%, more preferablyat least 60%, more preferably at least 70%, more preferably at least80%, more preferably at least 90%, more preferably at least 95%.

The invention relates to variants and fragments of above which have atleast one functional activity of a sequence selected from SEQ IDNOs:1-64. Thus, preferred variants and fragments of the invention arefunctional equivalents/homologues of the amino acid sequences identifiedas SEQ ID NOs:1-64.

Thus, by the term “functional equivalent/homologue” in the presentcontext is an amino acid sequence which has at least 65% of homologywith a sequences selected from SEQ ID NOs:1-64 or a sequence which hasat least 40% of the length of a sequence selected from SEQ ID NOs:1-64,and which is capable of at least one functional activity of the sequencewhich it has homology with, or which it is a fragment of, for example itis capable of stimulating neural plasticity, such as associated withneural cell differentiation and/or such as associated with memory andlearning, capable of stimulating of cell survival, such as inhibitingapoptosis, capable of binding to a receptor and modulating activity ofsaid receptor, or capable of inhibiting inflammation.

The invention relates both to naturally occurring, synthetically orrecombinantly prepared peptides and peptides prepared by means ofenzymatic/chemical cleavage of proteins. The peptides having the aminoacid sequences corresponding to subsequences of bigger polypeptides,such as for example peptides comprising or consisting of subsequences ofthe MTs described above, to be understood are derived from the sequencesof said bigger polypeptides or proteins. These peptides may be producedeither by enzymatic cleavage of proteins or prepared by means ofrecombinant expression or chemical synthesis.

Thus, the invention further relates to fragment of a metallothionein,said fragment preferably is derived from a human metallothionein, morepreferably from metallothionein-1 A (MT1A), metallothionein-1B (MT1B),metallothionein-1E (MT1E), metallothionein-1F (MT1F), metallothionein-1G(MT1G), metallothionein-1H (MT1H), metallothionein-1I (MT1I),metallothionein-1 K (MT1K), metallothionein-1 L (MT1L),metallothionein-1R (MT1R), metallothionein-1X (MT1 X), metallothionein-2(MT2), metallothionein-3 (MT3) or metallothionein-4 (MT4). The inventionrelates to a fragment of metallothionein which is capable of stimulatingneurite outgrowth, neuronal survival, activating a receptor, stimulatingneural plasticity associated with learning and memory and/or inhibitinginflammation. In particular, the invention relates to fragments of theabove mentioned metallothioneins which comprise or consist of a sequenceselected from SEQ ID NOs:9-64 or a fragments of said sequence. Inparticular embodiments a peptide fragment which comprises or consists ofa particular sequence of the described above may be preferred. Theinvention preferably relates to the fragments of metallothioneins whichcomprise at most 20 amino acids residues.

2. Compound

A compound may contain a single copy of an individual amino acidsequence selected from any of the described above, or it may contain twoor more copies of such amino acid sequence. This means that compound ofthe invention may be formulated as a monomer of a peptide sequence, suchas containing a single individual peptide sequence, or it may beformulated as a multimer of a peptide sequence, i.e containing two ormore individual peptide sequences, wherein said individual peptidesequences may be represented by two or more copies of the same sequenceor by two or more different individual peptide sequences. A multimer mayalso comprises a combination of the full-length sequence and one or morefragments thereof. In one embodiment a compound may contain two aminoacid sequences, such compound is defined herein as dimer, in anotherembodiment a compound may contain more then two amino acid sequences,such for example three, four or more sequences. The present inventionpreferably relates to compounds containing two or four peptide sequencesof the invention. However, compounds containing 3, 5, 6, 7, 8 or moresequences are also in the scope of the invention.

The compounds may be formulated as dimers or multimers comprising morethen two copies of individual peptide fragments which may have theidentical amino acid sequences or different amino acid sequences. Oneexample of such compound may be a dimeric compound containing SEQ ID NO:1 and SEQ ID NO: 2 or a dimeric compound containing SEQ ID NO: 1 and SEQID NO: 3. Any other combinations of the sequences of the invention maybe made depending on different embodiments. The sequences may beconnected to each other via peptide bond, or connected to each otherthrough a linker molecule or grouping.

As already mentioned above, a compound of the invention may contain twoor more copies of a single sequence, such as for example two copies ofany of the sequences selected from SEQ ID NOs: 1-64, wherein said twosequences may be connected to each other via a linker molecule orgrouping. A compound wherein the sequences are connected via a linkergrouping is preferred. One example of such linking grouping may be anachiral di-, tri- or tetracarboxylic acid. Suitable achiral di-, tri- ortetracarboxylic acids and a method of production such a compound (aligand presentation assembly method (LPA)) are described in WO0018791and WO2005014623. Another example of a possible linker may be the aminoacid lysine. Individual peptide sequences may be attached to a coremolecule such as lysine forming thereby a dendritic multimer (dendrimer)of an individual peptide sequence(s). Production of dendrimers is alsowell known in the art (PCT/US90/02039, Lu et al., (1991) Mol Immunol.28:623-630; Defoort et al., (1992) Int J Pept Prot Res. 40:214-221;Drijfhout et al. (1991) Int J Pept Prot Res. 37:27-32), and dedrimersare at present widely used in research and in medical applications. Itis a preferred embodiment of the invention to provide a dendrimericcompound comprising four individual amino acid sequences attached to thelysine core molecule. It is also preferred that at least one of the fourindividual amino acid sequences comprises an amino acid sequence of theformula defined above. It is even more preferred if the all fourindividual amino acid sequences of a dendrimeric compound individuallycomprise an amino acid sequence of the formula defined above.

Multimeric compounds of the invention, such as LPA-dimers orLysin-dendrmers, are among preferred compounds of the invention.However, other types of multimeric compounds comprising two or moreindividual sequences of the invention may be preferred depending on theembodiments.

3. Biological Activity

A peptide sequence of the invention and a compound comprising a sequenceof the invention possess biological activity. The invention preferablyrelates to a biological activity selected from

-   -   capability of stem cell differentiation, for example by        stimulating neuronal cell precursor differentiation;    -   capability of stimulating neural cell differentiation and/or        regeneration of nerves, for example by stimulating neurite        outgrowth;    -   capability of stimulating neural plasticity associated with        memory and learning, for example by stimulating synaptic        efficacy;    -   capability of stimulating of cell survival, in particular        stimulating survival neuronal cells, for example by inhibiting        apotosis of neural cells,    -   capability of inhibiting the oxidative stress response, for        example by stimulating expression of scavenges of reactive        oxygen species    -   capability to activate astrogliosis, such as capability of        stimulating astrocytes to express neuroprotective growth factors        and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin,        NGF, variety of fibroblast growth factors (FGFs), S100-proteins        (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin;    -   capability of inhibiting inflammation, for example inhibiting        the activation of microglia and macrophages, inhibiting        expression of pro-inflammatory cytokines and/or by stimulating        anti-inflammatory responses;    -   capability of stimulating angiogenesis in the lesioned area, for        example by stimulating expression of growth factor promoting        angiogenesis such as VEGF and FGF2.    -   capability of binding to a receptor, for example megalin        receptor (Swiss-prot Ass. number: P98164) or MT protein, and        accelerating transcytosis across the blood-brain barrier via        megalin receptor and/or modulating activity of said receptor,        for example activating or inhibiting signal transduction        associated with this receptor, or activating or inhibiting        biological function of the receptor;    -   capability of binding to a metallothionein and enhancing its        neuroprotective and neuroregenerative functions.

According to the invention, biological activities of the peptide whichare associated with physiological processes occurring in the brain orassociated with normal or pathological conditions of the neural systemare preferred. The molecular processes involving a biological activityof the peptide are preferably those that may be related to a cell of theneural system, more preferably to a neuronal cell. Accordingly, one ofthe preferred activities of the peptide according to the invention isthe capability of stimulating neuronal cell differentiation.

The term “neuronal differentiation” is understood herein both asdifferentiation of neural precursor cells, or neural stem cells, andfurther differentiation of neural cells, such as for example maturationof neuronal cells. An example of such differentiation may be neuriteoutgrowth from immature neurons, branching of neurites, and also neuronregeneration.

Thus, one preferred embodiment the invention concerns biologicalactivity of a peptide sequence associated with stimulating ofdifferentiation of neural precursor/stem cells or immature neuronsand/or stimulating neurite outgrowth from mature neurons, for examplesneurons which were traumatizes but survived and are committed toregenerate damaged processes.

In the present context “differentiation” is related to the processes ofmaturation of neurons and extension of neurites, which take place afterthe last cell division of said neurons. The compounds of the presentinvention may be capable of stopping neural cell division and initiatingmaturation said cells, such as initiating extension of neurites.Otherwise, “differentiation” is related to initiation of the process ofgenetic, biochemical, morphological and physiological transformation ofneuronal progenitor cells, immature neural cells or embryonic stem cellsleading to formation of cells having functional characteristics ofnormal neuronal cell as such characteristics are defined in the art. Theinvention defines “immature neural cell” as a cell that has at least onefeature of neural cell accepted in the art as a feature characteristicfor the neural cell.

Substances with the potential to promote neurite outgrowth as well asstimulate regeneration and/or differentiation of neuronal cells, such ascertain endogenous trophic factors, are prime targets in the search forcompounds that facilitate for example neuronal regeneration and otherforms of neuronal plasticity. To evaluate the potential of the presentcompound, the ability to stimulate the neurite outgrowth relatedsignalling, interfere with cell adhesion, stimulate neurite outgrowth,regeneration of nerves, may be investigated. Compounds of the presentinvention are shown to promote neurite outgrowth and are thereforeconsidered to be good promoters of regeneration of neuronal connections,and thereby of functional recovery after damages as well as promoters ofneuronal function in other conditions where such effect is required.

According to the present invention a compound comprising at least one ofthe above peptide sequences is capable of stimulating neurite outgrowth.The invention concerns the neurite outgrowth improvement/stimulationsuch as about 75% improvement/stimulation above the value of neuriteoutgrowth of control/non-stimulated cells, for example 50%, such asabout 150%, for example 100%, such as about 250, for example 200%, suchas about 350%, for example 300%, such as about 450%, for example 400%,such as about 500%.

Estimation of capability of a candidate compound to stimulate neuriteoutgrowth may be done by using any known method or assay for estimationof neurite outgrowth, such as for example as the described in Examplesbelow.

According to the invention a compound has neuritogenic activity both asan insoluble immobile component of cell growth substrate and as asoluble component of cell growth media. In the present context“immobile” means that the compound is bound/attached to a substancewhich is insoluble in water or a water solution and thereby it becomesinsoluble in such solution as well. For medical applications bothinsoluble and soluble compounds are considered by the application,however soluble compounds are preferred. Under “soluble compound” isunderstood a compound, which is soluble in water or a water solution.

Accordingly, the invention also concerns a method for stimulatingneuronal cell differentiation comprising using a peptide sequence of theinvention or a compound comprising said sequence.

One of most preferred embodiments of the invention concerns the activityof the peptide sequences in connection with learning and memory, inparticular, the capability of a peptide sequence to stimulate synapticplasticity, spine formation, synaptic efficacy. Thus, the invention alsoconcerns a method for stimulating memory and/or learning comprisingusing a peptide sequence of the invention and/or compound comprisingsaid sequence. The invention relates to both short-term memory andlong-term memory.

In another preferred embodiment of the invention a peptide sequence ofthe invention capable of stimulating cell survival, in particularneuronal cell survival. The invention concerns the capability ofstimulating cell survival both due trauma and degenerative disease.Accordingly, the invention relates to a method for stimulating cellsurvival, preferably neuronal cell survival by using a peptide sequenceof the invention and/or compound comprising said sequence.

Substances with the potential to enhance neuronal cells to survive dueto damage as well as inhibit degeneration and/or apoptosis of neuronalcells in trauma and disease, are prime targets in the search forcandidate compounds for new medicine for treatment of neurodegenerativediseases such as for example Alzheimer's or Parkinson's diseases. Toevaluate the potential of the present peptides, the ability to stimulatesurvival related signalling, interfere with apoptosis related cellularreactions, stimulate regeneration of nerves may be investigated.Compounds of the present invention are shown to promote neural cellsurvival and decrease the cell loss and therefore considered to be goodcandidates for promotion of regeneration of neural connections in brainand/or in peripheral neural system, and thereby of functional recoveryafter damages due trauma or disease as well as promoters of neuronalfunction in any other conditions where such effect is required.

In the present context “survival” is related to the processes associatedwith maintenance and/or recovery of cell function after the damage ofthe cell. The compounds of the present invention may be capable ofstopping or attenuating the processes committing the cell to death, suchas inhibiting apoptosis of neural cells initiated by cell damage duetrauma or disease. Otherwise, “survival” is related to inhibition of theprocesses associated with the cell damage leading to cell death andinitiation of the processes of genetic, biochemical, morphological andphysiological transformation or reconstruction of cells, in particularneuronal cells, such as progenitor cells, immature neural cells orembryonic stem cells or mature neural cells having normal functionalcharacteristics defined in the art. The invention defines “immatureneural cell” as a cell that has at least one feature of neural cellaccepted in the art as a feature characteristic for the neural cell.

According to the present invention a compound comprising at least one ofthe above peptide sequences is capable of stimulating neural cellsurvival. The invention concerns the neural cell survival stimulationsuch as about 75% stimulation above the value of survival ofcontrol/non-stimulated cells, for example 50%, such as about 150%, forexample 100%, such as about 250, for example 200%, such as about 350%,for example 300%, such as about 450%, for example 400%, such as about500%.

Estimation of capability of a candidate compound to stimulate neuralcell survival may be done by using any known method or assay forestimation of cell survival, such as for example the ones described inExamples of the present application.

According to the invention a compound has survival promoting activityboth as insoluble and soluble compound. In the present context“insoluble” means that the compound is bound/attached to a substancewhich is insoluble in water or a water solution and thereby the compoundbecomes insoluble in such solution as well. For medical applicationsboth insoluble and soluble compounds are considered by the application,however soluble compounds are preferred. Under “soluble compound” isunderstood a compound, which is soluble in water or a water solution.

In another embodiment the peptide sequence of the invention is alsocapable of inhibiting an inflammatory process, in particular aninflammatory process in the brain.

Inflammation is a defense reaction caused by tissue damage due to amechanical injury or bacterial, virus or other organism infection. Theinflammatory response involves three major stages: first, dilation ofcapillaries to increase blood flow; second, microvascular structuralchanges and escape of plasma proteins from the bloodstream; and third,leukocyte transmigration through endothelium and accumulation at thesite of injury and infection. The inflammatory response begins with arelease of inflammatory mediators. Inflammatory mediators are soluble,diffusible molecules that act locally at the site of tissue damage andinfection, and at more distant sites, influencing consequent events ofthe inflammatory response. Inflammatory mediators can be exogenous, e.g. bacterial products or toxins, or endogenous, which are producedwithin the immune system itself, as well as injured tissue cells,lymphocytes, mast cells and blood proteins.

Neuroinflammation plays a prominent role in the progression ofAlzheimer's disease and may be responsible for degeneration invulnerable regions such as the hippocampus. Neuroinflammation isassociated with elevated levels of extracellular glutamate andpotentially an enhanced stimulation of glutamate N-methyl-D-aspartatereceptors.

Anti-inflammatory activity is another important biological activity ofthe peptide sequence of the invention. Thus, the invention relates toanti-inflammatory peptide, which is capable of serving as an inhibitorof the sustained inflammatory response, in particular in the brain.

The continuous presence of inflammatory mediators, such as for exampleTNF alpha in the body in response to sustained presence of bacterialproducts or even live bacteria locally during days or weeks followingtrauma and/or infection promotes the reactions to inflammation, such as,for example, heat, swelling, and pain. The sustained inflammatoryresponse has been proven to be very harmful to the body. If thebacterial products or live bacteria become spread universally in thebody from their local focus the inflammatory reaction becomesoverwhelming and out of control and leads to sepsis which eventuallyprogress further to severe sepsis and septic shock. Anti-inflammatorypeptides may be used to block or suppress the overwhelming sustainedinflammatory response represented by a massive and harmful cytokinecascade in the blood and vital organs such as lung, liver intestine,brain and kidneys.

In the present context by the term “anti-inflammatory compound” is meanta compound which is capable of at least one of the following activities

-   i) decreasing or inhibiting the gene expression in the immune cells,    preferably monocytes/macrophages in response to bacterial products,    live bacteria or trauma to produce endogenous inflammatory mediators    including receptors for inflammatory mediators and transcription    factors involved in the signal transduction of the inflammatory    mediators, said mediators being preferably selected from the group    comprising cytokines, selected from the group TNFalpha IL-1, IL-6,    G-CSF, GM-CSF, M-CSF. Chemokines selected from the group comprising    IL-8, MCP-1, receptors selected from the group Tissue factor and    IL-2Ralpha,-   ii) decrease or inhibit the production bradykinin by the phase    contact system,-   iii) decrease or inhibit the attractant potential for monocytes,    and/or-   iv) decrease or inhibit the life-time of monocytes, neutrophils and    other immune cells serving as an inducer of apoptosis,-   v) decrease or inhibit vascular endothelial cells to express the    adhesion molecules, said adhesion molecules being preferably    selected from the group comprising PECAM, ICAM-1, E-selectins,    VCAM-1-   vi) decrease or inhibit activation of the contact phase system to    produce bradykinin leading to increased vascular permeability,-   vii) stimulate the synthesis of an anti-inflammatory mediator    selected from the group of IL-10 and IL-12,-   viii) inhibiting complement activation;-   ix) decreasing the risk of neural cell degeneration in the presence    of chronic neuroinflammation, e.g. neurons which express glutamate    N-methyl-D-aspartate receptors.

Another biological activity of a peptide of the invention which ispreferred among others is the capability of the peptide of to bind to areceptor.

In the present content the term “receptor” is defined as a functionalproteinaceous structure that tightly bind specific molecules (e.g. smallmolecules, proteins, viruses). The invention relates to receptors of theplasma membrane (surface) of cells, receptors which are located insidethe cell's plasma membrane, i.e. free-floating receptors, solubleproteininaceous molecules, i.e. carrier proteins, receptors locatedinside the cell and associated with different compartments of said cell,e.g. nucleus, mitochondria.

Both (membrane, internal) types of receptors are a functional part ofinformation transmission (i.e., signaling) to the cell. A generaloverview is that once bound, both the receptor and its “bound entity” asa complex is internalized by the cell via a process called endocytosis,in which the cell membrane in the vicinity of the bound complexinvaginates. This process forms a membrane “bubble” on the inside of thecell, which then pinches off to form an endocytic vesicle. The receptorthen is released from its bound entity by cleavage in the cell'slysosomes. It is recycled (returned) to the surface of the cell (e.g.,low-density lipoprotein receptors). In some cases the receptor, alongwith its bound molecule may be degraded by the powerful hydrolyticenzymes found in the cell's lysosomes (e.g., insulin receptors,epidermal growth factor receptors, and nerve growth factor receptors).Endocytosis (internalization of receptors and bound ligand such as ahormone) removes hormones from the circulation and makes the celltemporarily less responsive to them because of the decrease in thenumber of receptors on the surface of the cell. Hence the cell is ableto respond (to new signal).

The invention relates in a preferred embodiment to megallin receptor.The peptide sequence of the invention possesses a capability to bind tomegallin, and activate megallin associated signal transduction, oractivate signal transduction which is not directly associated withmegalin. The latter mode of “reception” occurs when, following binding,a transmembrane protein (e.g., megalin) activates the portion of thetransmembrane (i.e., through the cell membrane) protein lying inside thecell. That “activation” causes an effector inside cell to produce a“signal” chemical inside the cell which causes the cell's nucleus (viagene expression) to react to the original external chemical signal (thatbound itself to the receptor portion of the transmembrane protein).

A peptide sequence of the invention has a capability to bind to megalin.As megalin is a scavenger receptor and has a plethora of ligands, thepeptide sequence of the invention may competitively inhibit binding ofthe ligands to megalin and thus modulate the activity of said receptorassociated with said ligands.

Another type of receptor of the sequence of the invention is soluble MTprotein. A peptide sequence with is derived from a homophylic bindingsite of MT has a capability to bind to this homophylic site andtherefore affect homophylic binding of one or more molecules of MT. Thismay have a major influence on function of MT which is of importance fora huge number of the body physiological processes. Accordingly, theinvention relates to capability of binding a peptide sequence of theinvention to MT as a preferable biological activity of the sequence aswell.

4. Production of Individual Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the DNA sequences encoding the correspondingfull-length protein of peptide origin, using DNAase I according to astandard protocol (Sambrook et al., Molecular cloning: A Laboratorymanual. 2 rd ed., CSHL Press, Cold Spring Harbor, N.Y., 1989). Thepresent invention relates to full-length proteins selected from thegroups of proteins identified above. The DNA encoding the full-lengthproteins of the invention may alternatively be fragmented using specificrestriction endonucleases. The fragments of DNA are further purifiedusing standard procedures described in Sambrook et al., Molecularcloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor,N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983, Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al, eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4c (Russell et al., 1983, Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-S-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteins. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

By SAPS peptides may be synthesised either batchwise in a polyethylenevessel equipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A.and Sheppard, R. C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137.) ona fully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality.

When synthesised, individual peptide sequences may then be formulated asmultimers using well-known in the art techniques, for examples dimers ofthe sequences may be obtained by the LPA method described in WO00/18791, denrimeric polymers by the MAP synthesis described inPCT/US90/02039.

5. Antibody

It is another objective of the present invention to provide an antibody,antigen binding fragment or recombinant protein thereof capable ofrecognizing and selectively binding to an epitope comprising the motifof the invention or a sequence selected from SEQ ID NOs:1-64, or afragment of said sequence, preferably the epitope located within thesequence of MT protein, for example metallothionein-1 A (MT1A),metallothionein-1B (MT1B), metallothionein-1E (MT1E), metallothionein-1F(MT1F), metallothionein-1G (MT1G), metallothionein-1H (MT1H),metallothionein-11 (MT1I), metallothionein-1 K (MT1K), metallothionein-1L (MT1L), metallothionein-1R (MT1R), metallothionein-1X (MT1 X),metallothionein-2 (MT2), metallothionein-3 (MT3) or metallothionein-4(MT4)

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies(thereby causing an immune response). The term “epitope” is theequivalent to the term “antigenic determinant”. The epitope may comprise3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 aminoacid residues, located in close proximity, such as within a contiguousamino acid sequence, or located in distant parts of the amino acidsequence of an antigen, but due to protein folding have been approachedto each other.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA. 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called alpha (α), delta (β),epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains ofantibodies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody which includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody”, as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In preferred embodiments, in thecontext of both the therapeutic and screening methods described below,an antibody or fragment thereof is used that is immunospecific for anantigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising a peptide sequence selected from any of the sequencesidentified herein as SEQ ID NOs: 1-85, or a fragment of said sequences.Thus, in context of the present invention the term “antibody fragment”is identical to term “antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab′ fragments held together by        two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

-   -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule. Such single chain antibodies are also referred to as        “single-chain Fv” or “sFv” antibody fragments. Generally, the Fv        polypeptide further comprises a polypeptide linker between the        VH and VL domains that enables the sFv to form the desired        structure for antigen binding. For a review of sFv see Pluckthun        in The Pharmacology of Monoclonal Antibodies 113: 269-315        Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad Sci. USA 90: 6444-6448 (1993).

The invention contemplate both polyclonal and monoclonal antibody,antigen binding fragments and recombinant proteins thereof which arecapable of binding an epitope according to the invention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726, Cold Spring Harbor Pub. (1988), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, NY.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991,Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Biol 222:581-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., 1991, In: Methods: ACompanion to Methods in Enzymology, 2:97; Bird et al., 1988, Science242:423-426; U.S. Pat. No. 4,946,778; and Pack, et al., 1993,BioTechnology 11:1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the eitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising the epitope(s) described herein, is one of the preferredembodiments of the invention.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard method inthe art for producing polyclonal and monoclonal antibodies using naturalor recombinant fragments of human MT protein, such as metallothionein-1A (MT1A), metallothionein-1B (MT1B), metallothionein-1E (MT1E),metallothionein-1F (MT1F), metallothionein-1G (MT1G), metallothionein-1H(MT1H), metallothionein-11 (MT1I), metallothionein-1 K (MT1K),metallothionein-1 L (MT1L), metallothionein-1R (MT1R),metallothionein-1X (MT1 X), metallothionein-2 (MT2), metallothionein-3(MT3) or metallothionein-4 (MT4), said fragment comprising a structuralmotif of the invention, for example comprising a sequence selected fromSEQ ID NOs: 9-64, such as for example a sequences selected from SEQ IDNOs: 1-64, as an antigen. Such antibodies may be also generated usingvariants, homologues or fragments of peptide sequences of SEQ IDNOs:1-64 said variants, homologues and fragments are immunogenic peptidesequences which meet the following criteria:

-   (i) being a contiguous amino acid sequence of at least 6 amino    acids;-   (ii) comprising the motif of the invention.

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of animmunogenic fragment described above.

The invention relates both to antibodies which are capable ofmodulating, such as enhancing or attenuating, biological function ofmetallothionein-1 A (MT1A), metallothionein-1B (MT1B),metallothionein-1E (MT1E), metallothionein-1F (MT1F), metallothionein-1G(MT1G), metallothionein-1H (MT1H), metallothionein-11 (MT1I),metallothionein-1 K (MT1K), metallothionein-1 L (MT1L),metallothionein-1R (MT1R), metallothionein-1X (MT1 X), metallothionein-2(MT2), metallothionein-3 (MT3) or metallothionein-4 (MT4), and toantibodies which do not modulate the function of MT upon binding to theepitope of the invention. Preferred biological functions ofmetallothionein-1 A (MT1A), metallothionein-1B (MT1B),metallothionein-1E (MT1E), metallothionein-1F (MT1F), metallothionein-1G(MT1G), metallothionein-1H (MT1H), metallothionein-1I (MT1I),metallothionein-1 K (MT1K), metallothionein-1 L (MT1L),metallothionein-1R (MT1R), metallothionein-1X (MT1 X), metallothionein-2(MT2), metallothionein-3 (MT3) or metallothionein-4 (MT4)2 in thepresent context may be a capability of stimulating cell differentiationor cell survival, promoting nerve regeneration, inhibiting oxidativestress responses, activating astrocytes and their production of survivalpromoting growth factors and proteins, inhibiting inflammation,promoting morphological and functional plasticity of neural cells, e.g.enhancing synaptic plasticity promoting the formation of new functionalsynapses by newborn neurons.

6. Kit

The present invention discloses compounds that are capable of binding toMT. In one embodiment the invention relates to peptide sequencescomprising the amino acid motif described above or comprising orconsisting of a fragment of MT as described above. In another embodimentthe invention relates to anti-MT antibodies capable of binding to theepitope described above. Both peptide sequences and antibody may be usedfor detection of MT in sample collected from an individual, for examplea sample of a body fluid or biopsy.

In one aspect there is provided a kit for diagnosis of or for predictingthe risk of a subject for developing a disease associated with MTfunction. Non-limited examples of use such a kit may be estimation of anincreased risk for progression of primary melanoma (MT overexpression ina variety of cancers is associated with resistance to anticancer drugsand radiotherapy, and with a poor prognosis such diseases (Weinlich etal., Br J Dermatol. 2003, 149(3):535-41)), estimation of severity ofchronic hepatitis C virus infection (HCV) (MT expression could reflectthe severity of chronic HCV infection and could be one of the factorsassociated with a favorable clinical outcome in the response tointerferon therapy (Carrera et al., Am J Gastroenterol. 2003,98(5):1142-9)).

Thus, the invention a further embodiment relates to a diagnostic methodcomprising using a kit comprising a peptide sequence of the invention,compound of the invention and/or antibody of the invention. Thediagnostic method is to be used for diagnosis of a disease wherein MTpays a role in pathology. The kit according to the invention may be usedfor detection of MT in a sample collected from an individual.

The sample used in the diagnostic method of the invention is anybiological sample, such as for example a sample of blood, urine, body ortumor tissue, or any other appropriate biological sample.

In another embodiment there is provided a kit for inhibitingpolymerization of MT in solution (MT aggregation is a serious problemand results in overestimation of the protein in patient samples (Tang etal. J Anal Toxicol. 1999 May-June; 23(3):153-8), polymerization ofmetallothioneins is also one of the usually encountered puzzles duringthe research process of metallothioneins' structure and function (Hou etal. Protein Sci. 2000 9(12):2302-12)).

A kit according of the invention preferably comprises at least onepeptide sequence capable of binding to MT and/or at least one antibodyor an antigen-binding fragment thereof, wherein said peptide sequenceand/or said antibody or antibody fragment is linked to a detectablelabel.

The detectable label may be any type of label selected according to thedetection method to be used. An appropriate label may be selected fromany commercially available labels and used for labelling of any part ofthe kit of the invention, i.e. a peptide sequence, compound or/andantibody, according to the manufacturer instructions.

7. Pharmaceutical Composition

The invention also relates to a pharmaceutical composition comprisingone or more of the compounds defined above, wherein the compound iscapable of stimulating neurite outgrowth and/or neural celldifferentiation, survival of neural cells and/or stimulating learningand/or memory. Thus, the invention concerns a pharmaceutical compositioncapable of stimulating differentiation of neuronal cells and/orstimulating regeneration of neuronal cells, and/or stimulating neuronalplasticity in connection with learning and memory, and/or stimulatingsurvival of neural cells.

In the present context the term “pharmaceutical composition” is usedsynonymously with the term “medicament”.

In a composition the peptide sequences may be formulated as comprisingisolated individual peptide fragments or multimers or dimers thereof asdiscussed above.

The pharmaceutical composition may have the described above effects oncells in vitro or in vivo, wherein the composition is administered to asubject.

The medicament of the invention comprises an effective amount of one ormore of the compounds as defined above, or a composition as definedabove in combination with the pharmaceutically acceptable additives.Such medicament may suitably be formulated for oral, percutaneous,intramuscular, intravenous, intracranial, intrathecal,intracerebroventricular, intranasal or pulmonal administration.

Strategies in formulation development of medicaments and compositionsbased on the compounds of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude polyalkylene glycols or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient(s) in the range offrom 0.5% to 10%, preferably 1-2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and generally contain 10-95% of theactive ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaladministration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide compound) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic acid, oxalic acid, tartaric acid,mandelic acid, and the like. Salts formed with the free carboxyl groupmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred μg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 μg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. It is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecompounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

As discussed above, the present invention relates to treatment ofindividuals for inducing differentiation, stimulating regeneration,plasticity and survival of neural cells in vitro or in vivo, saidtreatment involving administering an effective amount of one or morecompounds as defined above.

Another strategy for administration is to implant or inject cellscapable of expressing and secreting the compound in question. Therebythe compound may be produced at the location where it is going to act.

8. Treatment

Brain disorders like traumatic injury, pellagra dementia/toxicity;epilepsy, brain ischemic/stroke; EAE/MS (multiple sclerosis); andinfectious encephalopathies; Amyotrophic Lateral Sclerosis, Parkinson'sdisease; peripheral nerve injury, cerebral malaria, ageing/age dementia,neuromuscular damage and diabetes, all, were associated with activity ofMT. The MT roles were partially or fully validated in human tissue orhuman patients during degenerative diseases such as AD, Pick's diseaseand ALS; and during MS, Binswanger's encephalopathy, and ischaemia andduring neuromuscular damage and diabetes. Also, during a number ofcommon autoimmune, inflammatory and allergic diseases.

Thus, in a further aspect, the present invention relates to the abovedescribed peptides, fragments, or variants thereof, compounds andantibodies as medicaments for treatment of diseases wherein their

capability of stem cell differentiation, for example by stimulatingneuronal cell precursor differentiation,

-   -   capability of stimulating neural cell differentiation and/or        regeneration of nerves, for example by stimulating neurite        outgrowth,    -   capability of stimulating neural plasticity associated with        memory and learning, for example by stimulating synaptic        efficacy,    -   capability of stimulating of cell survival, in particular        stimulating survival neuronal cells, for example by inhibiting        apotosis of neural cells,    -   capability of inhibiting the oxidative stress response, for        example by stimulating expression of scavenges of reactive        oxygen species,    -   capability to activate astrogliosis, such as capability of        stimulating astrocytes to express neuroprotective growth factors        and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin,        NGF, variety of fibroblast growth factors (FGFs), S100-proteins        (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin,    -   capability of inhibiting inflammation, for example inhibiting        the activation of microglia and macrophages, inhibiting        expression of pro-inflammatory cytokines and/or by stimulating        anti-inflammatory responses,    -   capability of stimulating angiogenesis in the lesioned area, for        example by stimulating expression of growth factor promoting        angiogenesis such as VEGF and FGF2,    -   capability of binding to a receptor, for example megalin        receptor (Swiss-prot Ass. number: P98164) or MT protein, and        accelerating transcytosis across the blood-brain barrier via        megalin receptor and/or modulating activity of said receptor,        for example activating or inhibiting signal transduction        associated with this receptor, or activating or inhibiting        biological function of the receptor,    -   capability of binding to a metallothionein and enhancing its        neuroprotective and neuroregenerative functions        may be essential for curing.

Treatment by the use of the compounds/compositions according to theinvention is in one embodiment useful for inducing differentiation,modulating proliferation, stimulate regeneration, neuronal plasticityand survival of cells being being resident or implanted or transplanted.

Thus, the treatment comprises treatment and/or prophylaxis of celldamage and/or cell death in relation to diseases or conditions of thecentral and peripheral nervous system, such as postoperative nervedamage, traumatic neuron damage, e.g. resulting from spinal cord injury,impaired myelination of nerve fibers, postischaemic damage, e.g.resulting from a stroke, multiinfarct dementia, multiple sclerosis,neuronal degeneration associated with diabetes mellitus, neuro-musculardegeneration, schizophrenia, Alzheimer's disease, Parkinson's disease,or Huntington's disease

Also, in relation to diseases or conditions of the muscles includingconditions with impaired function of neuro-muscular connections, such asgenetic or traumatic atrophic muscle disorders; or for the treatment ofdiseases or conditions of various organs, such as degenerativeconditions of the gonads, of the pancreas, such as diabetes mellitustype I and II, of the kidney, such as nephrosis the compounds accordingto the invention may be used for inducing differentiation, modulatingproliferation, stimulate regeneration, neuronal plasticity and survival,i.e. stimulating survival.

In yet a further embodiment the use of the compound and/orpharmaceutical composition is for the stimulation of the ability tolearn and/or of the short and/or long term memory.

In particular the compound and/or pharmaceutical composition of theinvention may be used in the treatment of clinical conditions, such aspsychoses, such as senile and presenile organic psychotic conditions,alcoholic psychoses, drug psychoses, transient organic psychoticconditions, Alzheimer's disease, cerebral lipidoses, epilepsy, generalparesis [syphilis], hepatolenticular degeneration, Huntington's chorea,Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of thebrain, syphilis, Schizophrenic disorders, affective psychoses, neuroticdisorders, personality disorders, including character neurosis,nonpsychotic personality disorder associated with organic brainsyndromes, paranoid personality disorder, fanatic personality, paranoidpersonality (disorder), paranoid traits, sexual deviations anddisorders, mental retardation, disease in the nerve system and senseorgans, cognitive anomalies, inflammatory disease of the central nervoussystem, such as meningitis, encephalitis, Cerebral degenerations such asAlzheimer's disease, Pick's disease, senile degeneration of brain,communicating hydrocephalus, obstructive hydrocephalus, Parkinson'sdisease including other extra pyramidal disease and abnormal movementdisorders, spinocerebellar disease, cerebellar ataxia, Marie's,Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellardegeneration, such as spinal muscular atrophy, familial, juvenile, adultspinal muscular atrophy, motor neuron disease, amyotrophic lateralsclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbarpalsy, primary lateral sclerosis, other anterior horn cell diseases,anterior horn cell disease, unspecified, other diseases of spinal cord,syringomyelia and syringobulbia, vascular myelopathies, acute infarctionof spinal cord (embolic) (nonembolic), arterial thrombosis of spinalcord, edema of spinal cord, subacute necrotic myelopathy, subacutecombined degeneration of spinal cord in diseases classified elsewhere,myelopathy, drug-induced, radiation-induced myelitis, disorders of theautonomic nervous system, disorders of peripheral autonomic,sympathetic, parasympathetic, or vegetative system, familialdysautonomia [Riley-Day syndrome], idiopathic peripheral autonomicneuropathy, carotid sinus syncope or syndrome, cervical sympatheticdystrophy or paralysis; peripheral autonomic neuropathy in disordersclassified elsewhere, amyloidosis, diseases of the peripheral nervesystem, brachial plexus lesions, cervical rib syndrome, costoclavicularsyndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachialneuritis or radiculitis, including in newborn. Inflammatory and toxicneuropathy, including acute infective polyneuritis, Guillain-Barresyndrome, Postinfectious polyneuritis, polyneuropathy in collagenvascular disease, disorders affecting multiple structures of eye,purulent endophthalmitis, diseases of the ear and mastoid process,abnormality of organs and soft tissues in newborn, including in thenerve system, complications of the administration of anesthetic or othersedation in labor and delivery, diseases in the skin includinginfection, insufficient circulation problem, injuries, including aftersurgery, crushing injury, burns. Injuries to nerves and spinal cord,including division of nerve, lesion in continuity (with or without openwound), traumatic neuroma (with or without open wound), traumatictransient paralysis (with or without open wound), accidental puncture orlaceration during medical procedure, injury to optic nerve and pathways,optic nerve injury, second cranial nerve, injury to optic chiasm, injuryto optic pathways, injury to visual cortex, unspecified blindness,injury to other cranial nerve(s), injury to other and unspecifiednerves. Poisoning by drugs, medicinal and biological substances, geneticor traumatic atrophic muscle disorders; or for the treatment of diseasesor conditions of various organs, such as degenerative conditions of thegonads, of the pancreas, such as diabetes mellitus type I and II, of thekidney, such as nephrosis, neoplasms such as malignant neoplasms, benignneoplasms, carcinoma in situ and neoplasms of uncertain behavior, morespecifically cancer in breast, thyroidal, pancreas, brain, lung, kidney,prostate, liver, heart, skin, blood organ (incl. but not limited to CMLand AML), muscles (sarcoma), cancers with dysfunction and/or over- orunder-expression of specific receptors and/or expression of mutatedreceptors or associated with soluble receptors, such as but not limitedto Erb-receptors and FGF-receptors; autoimmune disorders, such asrheumatoid arthritis, SLE, ALS and MS, anti-inflammatory effects,amyloidosis, chronic rheumatic heart disease, ischaemic heart disease,arrhythmia, asthma and other allergic reactions; diseases in thepulmonary system, respiratory system, sensoring e.g. oxygene. astma,complications of the administration of anesthetic or other sedation inlabor and delivery, acute myocardial infarction, and other relateddisorders or sequel from AMI; metabolic disorders, such as obscenitylipid disorders (e.g. hyper cholestorolamia, artheslerosis), diabetestype I and II, diseases of endocrine glands, such as diabetes mellitus Iand II, pituitary gland tumor, disorders of amino-acid transport andmetabolism, disorders of purine and pyrimidine metabolism and gout,myelopathy, drug-induced, radiation-induced, myelitis, bone disorders,such as fracture, osteoporosis, osteo arthritis (OA), obesity; stem cellprotection or maturation in vivo or in vitro, neurogenesis.

Inflammation of the brain is often consequence of infection, autoimmuneprocesses, toxins, and other conditions. Viral infections are arelatively frequent cause of this condition. Encephalitis may occur asprimary or secondary manifestation of TOGAVIRIDAE INFECTIONS;HERPESVIRIDAE INFECTIONS; ADENOVIRIDAE INFECTIONS; FLAVIVIRIDAEINFECTIONS; BUNYAVIRIDAE INFECTIONS; PICORNAVIRIDAE INFECTIONS;PARAMYXOVIRIDAE INFECTIONS; ORTHOMYXOVIRIDAE INFECTIONS; RETROVIRIDAEINFECTIONS; and ARENAVIRIDAE INFECTIONS.

Accordingly, a peptide, compound or a pharmaceutical composition of theinvention may be used for treatment inflammation in the brain associatedwith a viral infection.

A large body of clinical and experimental data indicate that complementactivation is an important mechanism for neuronal and glial injury inGuillain-Barré syndromes. Inhibition of complement activation thereforemight be expected to limit the progression of the disease (Halstead etal. (2005) Annals of Neurology 58:203-21).

Thus, in another embodiment, a peptide sequence, a compound andpharmaceutical composition may be used for treatment of Guillain-Barrésyndrome, its variant forms, such as Miller Fisher syndrome, and othercomplement dependent neuro-muscular disorders.

Peptide sequences, compounds and pharmaceutical composition may also beused for treatment children with autism.

Autism is a brain disorder that begins in early childhood and persiststhroughout adulthood; affects three crucial areas of development:communication, social interaction, and creative or imaginative play. Itis estimated to afflict between 2 and 5 of every 1000 children and isfour times more likely to strike boys than girls. Children with autismhave difficulties in social interaction and communication and may showrepetitive behaviour and have unusual attachments to objects orroutines.

In recent years, there have been scientific hints of immune systemirregularities in children with autism.

Thus, a peptide sequence, compound or a composition comprising thereofmay advantageously be used for treatment inflammation, in particularinflammation of the brain.

A further aspect of the invention is a process of producing apharmaceutical composition, comprising mixing an effective amount of oneor more of the compounds of the invention, or a pharmaceuticalcomposition according to the invention with one or more pharmaceuticallyacceptable additives or carriers, and administer an effective amount ofat least one of said compound, or said pharmaceutical composition to asubject.

In one embodiment of the process as mentioned above, the compounds areused in combination with a prosthetic device, wherein the device is aprosthetic nerve guide. Thus, in a further aspect, the present inventionrelates to a prosthetic nerve guide, characterised in that it comprisesone or more of the compounds or the pharmaceutical composition asdefined above. Nerve guides are known in the art.

Another aspect of the invention relates to the use of a compound asdefined above. In particular the use of a compound according to theinvention is for the production of a pharmaceutical composition. Thepharmaceutical composition is preferably for the treatment orprophylaxis of any of the diseases and conditions mentioned above.

In yet a further aspect the invention relates to a method of treating adisease or condition as discussed above by administering a compound asdefined herein.

EXAMPLES Peptides

MTAn: (SEQ ID NO: 1) KKSSCSCSPVGSAK MTAc: (SEQ ID NO: 2) AQGSISKGASDKSSMTBn: (SEQ ID NO: 3) MDPNSSSAAGDSST MTBc: (SEQ ID NO: 4) SAGSSKSKESKSTSMTAcc: (SEQ ID NO: 5) AQGSICKGASDKSS MTBnc: (SEQ ID NO: 6)MDPNCSCAAGDSST MTBcc: (SEQ ID NO: 7) SAGSCKCKESKSTS MT3Ip:(SEQ ID NO: 8) KGGEAAEAEAEK

Neuronal Differentiation Methods

Primary cultures of cerebellar granule neurons (CGN) from seven days oldrats were plated at a density of 10,000 cells/cm² in eight-well permanoxchamber slides and left to differentiate for 24 hours in the presence ofvarious concentrations of peptide. Cells were fixated and immuno-stainedfor GAP-43 to visualise only neurons. CGN cultures were fixed in 4%paraformaldehyde followed by blocking with 1% BSA and then incubatededwith polyclonal rabbit antibodies against rat GAP-43 (Chemicon, AHDiagnostics, Aarhus, Denmark) (1:1000 dilution with 1% BSA) followed byincubation with secondary Alexa Fluor®488 goat anti-rabbit antibodies(Molecular Probes, Eugene, Oreg., USA) (1:700 dilution with 1% BSA.Neurite length was estimated by means of a stereological approach (Rønnet al., 2000). Results are are expressed as percentage neurite outgrowth±SEM, with the untreated controls set at 100%. Statistics are performedwith a Student's paired t-test. *=p<0.05, **=p<0.01, ***=p<0.001. If notspecifically indicated, four experiments were performed for eachcondition. 2.

Results

Dissociated neurons from the cerebellum (CGN) were grown for 24 h in thepresence of the individual peptides. From FIGS. 1-5, it appears that thepeptides MTAc, MTBc, MTAcc, MTBnc and MTBcc stimulated differentiationof cerebellar primary neurons. The induction of neurite extension by allpeptides was dose-dependent with a bell-shaped curve. The effect of thepeptides MTBn and MTP31p was also tested, and it was found that thesepeptides had no neuritogenic activity.

Neuronal Survival In Vitro Methods 1. Cell Survival Assay

Primary CGN cultures from seven days old rats were plated at a densityof 100,000 cells/cm² in eight-well permanox chamber slides coated withpoly-D-Lysine and grown in a medium supplemented with 40 mM KCl. After24 hours cytosine-β-D-arabinofuranoside was added to the culture toinhibit proliferation of non-neuronal cells. Neurons were left todifferentiate for further 6 days in vitro, before apoptosis was inducedby shifting cells into a starving medium containing either 5 mM KClalone (negative control), 40 mM KCl (positive control) or 5 mM KCl plusvarious concentrations of peptide. 48 hours after apoptosis inductioncells were fixated and stained with Hoechst 33258. Neuronal viabilitywas estimated by comparing the amount of live neurons with the totalnumber of neurons based on nuclear morphology. Results (see FIGS. 6-9)from at least four independent experiments are expressed as percentagesurvival ±SEM, with the negative control set at 100%. Statistics areperformed with a Student's paired t-test. *=p<0.05, **=p<0.01,***=p<0.001.

2. TUNEL Assay (DNA Fragmentation)

Primary cultures of seven days old rats were plated, cultured andinduced to undergo apoptosis as described above for neuronal survival,however cells were fixated 24 hour after apoptosis induction. By usingthe Fluorescien FragEL DNA fragmentation Kit, which label free DNA endswith a green fluorescent colour, the amount of neurons undergoing DNAfragmentation can be estimated and compared to the total amount ofcells, which are stained with propidium idodide. Results (see FIG. 10)from at least four independent experiments are expressed as percentageTUNEL positive cells ±SEM, with the negative control set at 100%.Statistics are performed with a Student's paired t-test. *=p<0.05,**=p<0.01, ***=p<0.001.

Results

CGN from 7-day-old rats were induced to differentiate for 7 days in amedium with a high potassium concentration after which the neurons weregrown for two days in a low potassium medium either in the absence orpresence of IGF-1 (positive control), or the MT-derived peptides. Asappears from FIGS. 6-9, cell death induced by reducing the potassiumconcentration in the medium could be prevented by treatment with IGF-1.Moreover, treatment with the MT-derived peptides, MTAc, MTBc, MTBn,MTAcc, MTBcc, MTBnc and MT31p, all promoted survival of CGN in a dosedependent manner.

The anti-apoptotic effect of the MT-derived peptides was confirmed usinga DNA fragmentation assay. From FIG. 10 it appears that treatment withthe peptides MTAcc and MTBcc strongly reduced the number of apoptoticneurons.

Neuronal Rescue and Tissue Repair In Vivo Methods

A focal brain injury on the right fronto-parietal cortex was made byapplying a piece of dry-ice (−78° C.) directly onto the skull for 30seconds in mice and 60 seconds in rats, as previously described indetail (Penkowa and Moos, 1995). The rats were treated s.c. with thetetrameric form of the MTAcc peptide (AQGSICKGASDKSS) one day beforelesion and one and two days after the lesion (10 mg/kgbodyweight/injection). Three days after the lesion animals were fixed bytranscardial perfusion with paraformaldehyde. Histochemistry andimmunohistochemistry (IHC) were performed on sections cut from organstaken from fixated animals. For immunohistochemical investigation,brains were dissected and postfixed in Zamboni's fixative for 2-3 hours,dehydraded in graded alcohol followed by xylol and subsequently embeddedin paraffin before being cut in 3 μm frontal sections throughout theentire area of the lesion. Terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling(TUNEL) was performed using the Fragment End Labeling (FragEL™)Detection Kit (Calbiochem, USA, code QIA33). The FragEL kit contains allthe materials used below and each step was performed according to themanufacturer's recommendations. Sections were also immuno-stained formarkers of oxidative stress such as peroxynitrite-induced nitration oftyrosine residues (NITT) and malondialdehyde (MDA) andfor markers ofinflammation such as interleukin (IL)-1, IL-12 and tumor necrosis factor(TNF) a, as described by Penkowa et al. (2000).

Results

From FIG. 11 it can be seen that treatment of rats with the MTAccpeptide resulted in a dramatic decrease of the number of apoptotic cell(determined by TUNEL staining), inhibition of oxidative stress(determined by NITT and MDA immonostaining) and inhibition of theinflammatory response to the lesion (determined by immunostaining forinflammatory markers IL-1, IL-12 and TNFα).

Binding Peptides to MT2 Protein In Vitro Methods Surface PlasmonResonance (SPR) Analysis

Analysis of binding was performed employing a BIAcoreX instrument(Biosensor AB, Uppsala, Sweden) at 25° C. using 10 mM pH 7.4 sodiumphosphate containing 150 mM NaCl as running buffer (phosphate-bufferedsaline, PBS). The flow-rate was 5 μl/min. Data were analysed bynon-linear curve-fitting using the manufacturer's software. The MT2protein (from Sigma) was immobilized on a sensor chip CM5 using an aminecoupling kit (Biosensor AB) as follows: the chip was activated by 20 μlactivation solution; the protein was immobilized using 12 μl 20 μg/mlprotein in 10 mM sodium phosphate buffer pH 6.0; the chip was blocked by35 μl blocking solution. Various peptides at the indicatedconcentrations were injected into the sensor chip. The curvecorresponding to the difference between binding to MT2 and a blank chipwas used for analysis.

Results

It is known that MT can oligomerize in biological fluids (Tang et al.,1999), although the mechanism and the binding sites responsible forpolymerizaion are not known. By employing SPR we tested whether peptidesderived from MT can bind to the MT protein. From FIGS. 1, 2 and 3 itappears that the peptides MTAcc, MTBcc and MTAn specifically bind to theimmobilized on a sensor chip MT2 protein with the equilibriumdissociated constants (Kd) indicated in Table 1.

TABLE 1 Affinity binding constants k_(a) (M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (M)MT-2:MTAcc 4.54 ± 0.21 × 4.00 ± 0.27 × 8.95 ± 1.00 × 10³ 10⁻⁴ 10⁻⁸MT-2:MTBcc 2.83 ± 0.93 × 5.01 ± 1.75 × 1.60 ± 0.23 × 10³ 10⁻⁴ 10−⁷MT-2:MTAn 1.57 ± 0.05 × 5.74 ± 0.19 × 3.67 ± 0.16 × 10⁴ 10⁻³ 10⁻⁷

1.-36. (canceled)
 37. A method for stimulating neurite outgrowth, neuralcell survival, neural cell differentiation, neural plasticity associatedwith learning and memory, and/or inhibiting inflammation, comprising astep of administering a peptide of 13 to 17 amino acids in length,wherein the peptide is comprised of the amino acid sequenceSAGSCKCKESKSTS (SEQ ID NO:7) or a variant of SEQ ID NO:7, said varianthaving at least 65% sequence identity to SEQ ID NO:7.
 38. A method forstimulating neurite outgrowth, neural cell survival, neural celldifferentiation, neural plasticity associated with learning and memory,and/or inhibiting inflammation, comprising a step of administering thepeptide of claim 37, comprising an amino acid motif of the formula:S/D/E-(x)_(n)-S/D/E-K/S, wherein (x)_(n) is a sequence of any amino acidresidues, n is an integer from 4 to 5; and the peptide has no more thana total of six amino acid substitutions compared with SEQ ID NO:7.
 39. Amethod for stimulating neurite outgrowth, neural cell survival, neuralcell differentiation, neural plasticity associated with learning andmemory, and/or inhibiting inflammation, comprising a step ofadministering the peptide according to claim 38, wherein n is
 4. 40. Amethod for stimulating neurite outgrowth, neural cell survival, neuralcell differentiation, neural plasticity associated with learning andmemory, and/or inhibiting inflammation, comprising a step ofadministering the peptide according to claim 38, wherein n is
 5. 41. Amethod for stimulating neurite outgrowth, neural cell survival, neuralcell differentiation, neural plasticity associated with learning andmemory, and/or inhibiting inflammation, comprising a step ofadministering the peptide according to claim 38, wherein the amino acidsequence (x) comprises at least one of K, S, E, and C.
 42. A method forstimulating neurite outgrowth, neural cell survival, neural celldifferentiation, neural plasticity associated with learning and memory,and/or inhibiting inflammation, comprising a step of administering thepeptide according to claim 38, wherein said peptide comprises at leastone amino acid residue G within a sequence of 10 amino acid residuescomprising the motif.
 43. A method for stimulating neurite outgrowth,neural cell survival, neural cell differentiation, neural plasticityassociated with learning and memory, and/or inhibiting inflammation,comprising a step of administering the peptide according to claim 42,wherein said amino acid residue G immediately precedes an amino acidresidue S/D/E of said motif.
 44. A method for stimulating neuriteoutgrowth, neural cell survival, neural cell differentiation, neuralplasticity associated with learning and memory, and/or inhibitinginflammation, comprising a step of administering a peptide according toclaim 37, wherein said peptide consists of the amino acid sequence ofSEQ ID NO:7.
 45. A method for stimulating neurite outgrowth, neural cellsurvival, neural cell differentiation, neural plasticity associated withlearning and memory, and/or inhibiting inflammation, comprising a stepof administering a compound comprising one or more peptides, whereineach of said one or more peptides has a length of from 13 to 17 aminoacids, wherein each of said one or more peptides comprises the aminoacid sequence SAGSCKCKESKSTS (SEQ ID NO:7) or a variant of SEQ ID NO:7,said variant having at least 65% sequence identity to SEQ ID NO:7. 46.The method according to claim 45, wherein said compound comprising oneor more peptides consists of a single peptide.
 47. The method accordingto claim 45, wherein said compound comprising one or more peptidescomprises two or more of said peptide.
 48. The compound according toclaim 47 comprising a dimer or a tetramer of said peptide.
 49. Thecompound according to claim 47 comprising a dendrimer of said peptide.50. A method for stimulating neurite outgrowth, neural cell survival,neural cell differentiation, neural plasticity associated with learningand memory, and/or inhibiting inflammation, comprising a step ofadministering a pharmaceutical composition comprising a peptide having alength of from 13 to 17 amino acids, wherein the peptide comprises theamino acid sequence SAGSCKCKESKSTS (SEQ ID NO:7) or a variant of SEQ IDNO:7, said variant having at least 65% sequence identity to SEQ ID NO:7.51. A method for stimulating neurite outgrowth, neural cell survival,neural cell differentiation, neural plasticity associated with learningand memory, and/or inhibiting inflammation, comprising a step ofadministering a pharmaceutical composition comprising a peptide having alength of from 13 to 20 amino acids, wherein the peptide comprises theamino acid sequence SAGSCKCKESKSTS (SEQ ID NO:7) or a variant of SEQ IDNO:7, said variant having at least 65% sequence identity to SEQ ID NO:7.